The B lymphoma cell line Raji was treated with 10 μg/mL chimeric anti–HLA-DR antibody BK267, anti-CD20 antibody B1, or control antibody trastuzumab (TX). After 24 hours, cell death was evaluated (A) by 7-AAD staining and standard FACS analysis, (B) by incubation for 5 hours with alamarBlue vital dye, or (C) by performing 7-AAD staining followed by cytospin, fixing, and coloration of nuclei with DAPI. In panel C, photographs of cells stained with DAPI (left) or 7-AAD (right) are shown, as well as the percentage of 7-AAD⁺ cells with respect to total DAPI⁺ cells in the same experiment (table on the right). Control B-CLL cells treated with alemtuzumab and 20% human serum are shown in panel C. Finally the absolute number of cells scored by flow cytometry using reference beads was also determined by FACS (D). The data shown are the result of 1 representative experiment of at least 5 performed with 3 different cell lines. For panel C, slides were viewed with a Nikon Eclipse E800 microscope using a Nikon 20× Plan Fluor DICM lens at room temperature with Vectashield Medium (Vector Laboratories). Fluorochromes were CD19-FITC and 7-AAD (both BD Biosciences). Images were acquired using a Nikon Digital Sights DS-UI camera and were processed with Lucia Imaging Analysis Systems (Laboratory Imaging) and ImageJ software.